Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) has recently emerged as a promising therapeutic target in cancer. In this study, we explored the biological function of MAP4K4 in radioresistant breast cancer cells using two MAP4K4 inhibitors, namely PF06260933 and GNE-495. Radioresistant SR and MR cells were established by exposing SK-BR-3 and MCF-7 breast cancer cells to 48–70 Gy of radiation delivered at 4–5 Gy twice a week over 10 months. Surprisingly, although radioresistant cells were derived from two different subtypes of breast cancer cell lines, MAP4K4 was significantly elevated regardless of subtype. Inhibition of MAP4K4 with PF06260933 or GNE-495 selectively targeted radioresistant cells and improved the response to irradiation. Furthermore, MAP4K4 inhibitors induced apoptosis through the accumulation of DNA damage by inhibiting DNA repair systems in radioresistant cells. Notably, Inhibition of MAP4K4 suppressed the expressions of ACSL4, suggesting that MAP4K4 functioned as an upstream effector of ACSL4. This study is the first to report that MAP4K4 plays a crucial role in mediating the radioresistance of breast cancer by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis. We hope that our findings provide a basis for the development of new drugs targeting MAP4K4 to overcome radioresistance.

the inhibition of apoptosis 17,[23][24][25] .However, there is no information available on its involvement in resistance to standard cancer therapies.In this study, we discovered that MAP4K4 was highly overexpressed in radioresistant breast cancer cells.Consequently, we investigated the biological function of MAP4K4 in these cells using two MAP4K4 inhibitors, namely PF06260933 and GNE-495.

MAP4K4 was overexpressed in radioresistant breast cell lines derived from SK-BR-3 and MCF-7 cells
To establish radioresistant breast cancer cells, an epidermal growth factor receptor 2 (HER2)-positive SK-BR-3 and an estrogen receptor (ER)-positive MCF-7 cells were irradiated using cycles of 4 Gy or 5 Gy, respectively, twice weekly, which resulted in ~ 20% cell survival.Surviving cells were allowed to recover for 6-weeks between cycles and radiosensitivities were monitored.Cycles were continued until meaningful radioresistance had been established.As a result, radioresistant SK-BR-3 (SR) and MCF-7 (MR) cells were successfully established after 6 and 7 cycles, respectively, which resulted in cumulative doses of 48 Gy for SK-BR-3 cells and 70 Gy for MCF-7 cells.When SR and MR cells were irradiated with 1, 2, 3, 4, or 5 Gy and then subjected to clonogenic survival assays, they exhibited significant radioresistance as compared with parental cells (Fig. 1A).
To investigate the role of MAP4K4 in mediating radioresistance, we first compared endogenous MAP4K4 expression levels in parental and radioresistant cells.Interestingly, although SR and MR cells were derived from two different subtypes of breast cancer cell lines, MAP4K4 was similarly dysregulated regardless of subtype.Specifically, the protein expression of MAP4K4 was barely detectable in parental SK-BR-3 or MCF-7 cells but was significantly elevated in SR and MR cells (Fig. 1B).Furthermore, immunofluorescence staining demonstrated that MAP4K4 was mainly localized in cytoplasm in radioresistant cells but was non-detectable in parental cells (Fig. 1C).
As MAP4K4 is known to function through mitogen-activated protein kinase (MAPK) pathways, including MAPK/ERK1/2, MAPK/JNK 1/2, and MAPK/p38, we next checked whether the expressions of MAPKs were elevated in radioresistant cells.Surprisingly, the endogenous expression levels of p-ERK 1/2 and p-JNK 1/2 were found to be lower in SR and MR cells compared to parental cells (Fig. 1D), suggesting that JNK 1/2 and ERK1/2 are not regulated by MAP4K4 in radioresistant cells.However, the expression of p-p38 was significantly increased in SR and MR cells (Fig. 1D).

MAP4K4 inhibitors selectively targeted radioresistant breast cancer cells
To investigate the role of MAP4K4 in radioresistance in breast cancer cells, we utilized two MAP4K4 inhibitors, namely PF06260933 14 and GNE-495 26 .Given that MAP4K4 has kinase activity, we initially tested whether these MAP4K4 inhibitors could suppress its kinase activity.As anticipated, both PF06260933 and GNE-495 efficiently inhibited its kinase activity (Fig. 2A).However, to our surprise, western blot analysis revealed an unexpected result: while PF06260933 inhibited MAP4K4 protein expression, GNE-495 did not (Fig. 2B).
To assess the cytotoxic effects of PF06260933 and GNE-495 on SR and MR cells, parental and radioresistant cells were exposed to different concentrations of PF06260933 or GNE-495 for 48 h and then evaluated cell viability.Surprisingly, PF06260933 selectively targeted SR and MR cells, while leaving parental cells mostly unaffected (Fig. 2C).Specifically, at 40 μM concentration, SR and MR cells exhibited cell viabilities around 60% and 50%, respectively, whereas over 80% of parental cells survived (Fig. 2C).Similarly, GNE-495 exhibited greater cytotoxic effects on SR and MR cells compared to parental cells (Fig. 2D), indicating the importance of MAP4K4 kinase activity for the survival of radioresistant cells.

MAP4K4 inhibitors suppressed tumor growth in mice bearing SR tumors
As MAP4K4 inhibitors showed significant cytotoxic effects on SR and MR cells in vitro, we proceeded to evaluate the potential anti-tumor effects of PF06260933 in vivo.Previously, we reported that SR cells gained tumorigenic potential in vivo, whereas parental SKBR-3 cells were not able to form tumors in mice even when the number of injected cells was increased to 2 × 10 7 /mouse 27 .To assess the in vivo efficacy of PF06260933 on radioresistant breast cancer cells, we implanted SR cells into the mammary gland fat pads of female BALB/c nude mice, and when tumors became palpable, we intraperitoneally injected PBS or 10 mg/kg of PF06260933, three times a week for two weeks (Fig. 3A).Notably, the treatment schedules did not affect the body weight of the mice (Fig. 3B).As observed in vitro, PF06260933 treatment in vivo resulted in ~ 40% reduction in tumor growth compared to untreated mice (Fig. 3C and D).

MAP4K4 inhibitors induced apoptosis by suppressing DNA damage response
Next, we investigated the mechanisms underlying the cytotoxic effects of MAP4K4 inhibitors on radioresistant cells.In SR and MR cells, only the expression of p-p38 was upregulated (Fig. 1D).Thus, we first checked whether targeting MAP4K4 in radioresistant cells diminished p-p38 expression.Unexpectedly, inhibition of MAP4K4 with PF06260933 or GNE-495 did not inhibit p-p38 expression but instead induced its expression, along with p-H2AX expression (Fig. 4A).Since phosphorylations of H2A.X and p38 increase in response to DNA damage 28,29 , this observation indicated that p-p38 acts as a DNA damage marker rather than a downstream effector of MAP4K4 in radioresistant cells.Furthermore, PF06260933 and GNE-495 suppressed DNA repair response, as indicated by a reduction in RAD51 expression (Fig. 4A).These observations suggest that targeting MAP4K4 with PF06260933 or GNE-495 inhibits the activations of DNA repair systems, resulting in DNA damage accumulation.
As the accumulation of DNA damage often leads to apoptosis, we also explored whether MAP4K4 inhibitors induce apoptosis by evaluating the levels of cleaved caspase 3 and the fragmented form of poly [ADP-ribose] polymerase 1 (PARP), as PARP is cleaved into 89 and 24 kDa fragments by the cleaved form of caspases during apoptosis 30 .Treatment of SR cells with PF06260933 or GNE-495 significantly increased the cleaved form of caspase 3 and the fragmented form of PARP, while the level of the anti-apoptotic protein, survivin, was attenuated (Fig. 4B), suggesting that MAP4K4 regulates apoptosis.
These findings suggest that targeting MAP4K4 accumulates DNA damage by inhibiting the activation of DNA repair systems, and thus, induces the apoptosis of radioresistant cells.

MAP4K4 functioned as an upstream effector of ACSL4
In our previous study, we demonstrated that acyl-CoA synthetase-4 (ACSL4) contributes to radioresistance in breast cancer cells by enhancing DNA damage response and inhibiting apoptosis 27 .Since MAP4K4 also   contributes to radioresistance by regulating DNA damage response and apoptosis, we investigated whether there is an interaction between MAP4K4 and ACSL4 that mediates radioresistance.To explore this, we performed immunofluorescence staining to detect the localization of MAP4K4 and ACSL4 in radioresistant cells.We found that some MAP4K4 staining co-localized with ACSL4 staining in cytoplasm in SR and MR cells, but was undetectable in parental cells (Fig. 5A).Next, we examined the effects of inhibiting MAP4K4 or ACSL4 on the expression of the other protein.We observed that the inhibition of MAP4K4 with PF06260933 or GNE-495 reduced the expression of ACSL4 (Fig. 5B), while the inhibition of ACSL4 with triacsin C did not affect the expression of MAP4K4, as well as its kinase activity (Fig. 5C and D).These findings suggest that MAP4K4 acts upstream of ACSL4 in radioresistant cells.To confirm this, we performed siRNA transfection targeting MAP4K4 or ACSL4 in SR cells.Consistent with the results from the inhibitors, knockdown of MAP4K4 reduced the expression of ACSL4 (Fig. 5E), while knockdown of ACSL4 did not affect the expression of MAP4K4 (Fig. 5F).
Overall, our results indicate that there is an interaction between MAP4K4 and ACSL4 that mediates radioresistance in breast cancer cells, with MAP4K4 acting as an upstream effector of ACSL4.

Targeting MAP4K4 overcame the radioresistances of SR and MR cells
Encouraged by the observation that MAP4K4 inhibitors selectively targeted radioresistant cells, we investigated whether targeting MAP4K4 could overcome radioresistance in breast cancer.Typically, breast cancer patients receive 1.8-2 Gy of radiation 5 days a week for 5-6 weeks during conventional radiation therapy.Thus, we www.nature.com/scientificreports/treated SR, MR, and parental cells with 2 Gy daily for 5 days in the presence or absence of 20 μM PF06260933 or 500 nM GNE-495, and then performed clonogenic survival assays.As expected, SR and MR cells exhibited radioresistance under these conditions (Fig. 6A).About half of the SR and MR cells survived, whereas parental SK-BR-3 and MCF-7 cells were eradicated after exposure (Fig. 6A).However, when MAP4K4 was inhibited, the radioresistance of SR and MR cells were significantly reduced.The survival fractions of SR and MR cells irradiated at 2 Gy × 5 in the presence of PF06260933 were reduced by about 30-60% compared to cells irradiated without PF06260933 (Fig. 6B).Similarly, treatment with GNE-495 also reduced survival fractions of SR and MR cells (Fig. 6C).Additionally, a Transwell invasion assay showed that PF06260933 or GNE-495 efficiently suppressed SR and MR cell migration, while the majority of untreated SR and MR cells easily migrated through membranes (Fig. 6D).Taken together, these observations suggest targeting MAP4K4 can overcome radioresistance and inhibit the metastatic properties of radioresistant breast cancer cells.

Discussion
Although MAP4K4 has been identified as a promising target for cancer treatment 31 , its involvement in resistance to conventional cancer therapies remains unclear.This study provides evidence that targeting MAP4K4 can overcome radioresistance of breast cancer by downregulating ACSL4, which suppresses DNA damage response and ultimately induces apoptosis (Fig. 7).Breast cancers are highly heterogeneous due to variations in the expression of hormone receptors (estrogen and progesterone receptors) and HER2, which necessitates different treatment approaches, such as hormonal therapy, HER2 targeted therapy, or chemotherapy, based on molecular subtypes.Notably, the radioresistant breast cancer cell lines used in this study, SR and MR cells, were derived from two different breast cancer subtypes, HER2-positive SK-BR-3 and ERα-positive MCF-7 cells, respectively.Despite this difference, both radioresistant breast cancer cell lines exhibited a marked upregulation of MAP4K4 expression.Targeting MAP4K4 with two well-known inhibitors, PF06260933 14 and GNE-495 26 , improved the efficacy of radiotherapy in both radioresistant cancer cell lines.Interestingly, GNE-495 inhibited MAP4K4 kinase activity without affecting its protein expression, whereas PF06260933 inhibited both the kinase activity and protein expression.Nevertheless, both inhibitors overcame radioresistance in SR and MR cells, indicating that MAP4K4 kinase activity is critical for the survival of radioresistant cells.
To better understand the role of MAP4K4 in mediating radioresistance, it is important to identify its downstream mediators.Although it is known that MAP4K4 functions through MAPK pathways, including JNK 1/2, ERK 1/2, and p38, it remains unclear which of the MAPK pathways is activated by MAP4K4 in cancer.The activation of ERK 1/2 and p38 by MAP4K4 has been primarily reported in various biological processes other than cancer [31][32][33][34] .Concerning cancer, Gao et al. demonstrated that MAP4K4 activated ERK1/2 in lung adenocarcinoma 19 .In addition, some studies have shown that the activation of JNK 1/2 by MAP4K4 is associated with the motility of cancer cells and epithelial-mesenchymal transition (EMT) 17,23,25 .
In contrast to previous reports, in this study, we found that the levels of p-JNK 1/2 and p-ERK 1/2 were reduced in radioresistant SR and MR cells, while MAP4K4 levels were higher than those in parental cells.These findings indicate that neither JNK 1/2 nor ERK1/2 was affected by MAP4K4 in radioresistant cells.On the other hand, p-p38 expression was upregulated in radioresistant cells, but the inhibition of MAP4K4 with PF06260933 or GNE 495 did not inhibit p-p38 expression.Instead, the p-p38 level was induced along with that of p-H2AX.Since H2A.X and p38 are phosphorylated in response to DNA damage 28,29 , our data suggest that p38 acts as a DNA damage marker rather than a downstream effector of MAP4K4 in SR and MR cells.Taken together, these results suggest that MAP4K4 does not function through MAPK pathways to mediate radioresistance.
Instead of regulating MAPK pathways, we found that there is an interaction between MAP4K4 and ACSL4 that mediates radioresistance in breast cancer cells, with MAP4K4 acting as an upstream activator of ACSL4.Specifically, MAP4K4 inhibition with MAP4K4 inhibitors or siRNA efficiently suppressed the expression of ACSL4 in radioresistant cells, whereas ACSL4 inhibition by ACSL4 siRNA or triacsin C did not alter MAP4K4 expression.ACSL4 is one of five acyl-CoA synthetase long-chain family (ACSL) isoforms that convert long-chain fatty acids into the active form acyl-CoA and catalyze subsequent metabolism 35 .Recent studies have shown that ACSL4 is abnormally expressed in many types of cancer and is related to poor patient survival 36,37 .With regards to breast cancer, studies have reported that ACSL4 overexpression is associated with an aggressive breast cancer phenotype and promotes resistance to hormone therapy [38][39][40] .In our previous study, we found that ACSL4 enhances DNA damage response and inhibits apoptosis to mediate radioresistance by functioning an upstream effector of Forkhead box protein M1 (FOXM1) 27 .FOXM1 plays an essential role in the regulation of a wide spectrum of biological functions, including cell proliferation, cell cycle progression, cell differentiation, cell survival, and DNA damage repair in cancer cells [41][42][43] .However, the upstream regulator of ACSL4 remained unidentified.In this study, we identified MAP4K4 as an upstream effector of ACSL4 in radioresistant breast cancer cells.Thus, MAP4K4 contributes to radioresistance in breast cancer by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis.
Overall, our findings suggest that targeting MAP4K4 could be a promising therapeutic strategy for treating radioresistant breast cancer.We hope that this study provides a basis for the development of new drugs that target MAP4K4 to overcome radioresistance.

Cell culture
SK-BR-3 and MCF-7 human breast cancer cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in DMEM containing 10% fetal bovine serum and 1% antibiotic/antimycotic solution.Media for MCF-7 cells was also supplemented with insulin at 10 μg/mL.

Clonogenic survival assay
To confirm the acquisition of radioresistance, cells were irradiated with 1, 2, 3, 4, or 5 Gy once.To assess the effects of MAP4K4 inhibitors on radioresistance, cells were irradiated with 2 Gy per day for 5 days with or without 20 μM PF06260933 or 500 nM GNE-495.After culture for 10 days, colonies were fixed with 10% formalin and stained with 0.01% crystal violet.A colony was defined as a group of > 50 cells and colonies were counted under a microscope (TS 100, Nikon, Japan).Survival fractions were calculated by comparing the colony numbers of treated and non-treated control cells.

Chemosensitivity assay
Cells (1000-4000 cells/well) were treated with PF06260933 or GNE-495 for 48 h, fixed with 10% trichloroacetic acid, and stained with SRB for 30 min.After washing with 1% acetic acid to remove excess dye, 10 mM Tris base solution was added to each well to dissolved the protein-bound dye, and absorbances were measured at 510 nm using a microplate reader (Molecular Devices, CA, USA).

Immunofluorescence
SK-BR-3 and SR cells plated on chamber slides were incubated for 24 h, fixed with ice-cold methanol and acetone for 4 min and 2 min, respectively, blocked with 10% FBS, and incubated with a rabbit MAP4K4 antibody and a mouse ACSL4 antibody at 4 °C overnight.Slides were then washed with PBS and incubated with Alexa 488-conjugated goat anti-rabbit antibody and Alexa 546-conjugated goat anti-mouse antibody for 2 h in the dark.

Figure 3 .
Figure 3. MAP4K4 inhibitor suppressed tumor growth in mice bearing SR tumor.(A) The experimental scheme used for testing the anti-tumor effects of PF06260933 (PF) in vivo.(B) No loss of body weight occurred in mice treated with PF06260933.(C) Tumor growth in SR tumor-bearing BALB/c nude mice treated with PF06260933 (n = 4).(D) Final tumor volume on day of sacrifice.* indicates P value of < 0.05 versus control group.

Figure 5 .
Figure 5. MAP4K4 functioned as an upstream effector of ACSL4.(A) Immunofluorescence analysis of MAP4K4 and ACSL4 in SR and MR cells indicated that MAP4K4 co-localized with ACSL4 in radioresistant cells.(B) Expressions of ACSL4 in SR cells treated with PF06260933 or GNE-495.(C) Expressions of ACSL4 and MAP4K4 in SR cells treated with triacsin C (ACSL4 inhibitor).(D) Effect of triacsin C on MAP4K4 kinase activity.The protein expressions of MAP4K4 and ACSL4 following siRNA knockdown.MAP4K4 (E) and ACSL4 (F) protein levels are expressed as fold changes versus untreated cells (0 µM). Results are presented as means ± SDs, and * and ** indicate P values of < 0.05 and < 0.01, respectively.

Figure 6 .
Figure 6.Targeting MAP4K4 overcame the radioresistances of SR and MR cells and suppressed their metastatic properties.(A) Radioresistance of SR and MR cells after exposure to 2 Gy/day for 5 days.SR and MR cells were highly resistant to 2 Gy/day × 5 radiation as compared with parental cells.Clonogenic survival assays of SR and MR cells exposed to 2 Gy/day for 5 days in presence or absence of (B) PF06260933 (PF) or (C) GNE-495 (GNE).Results are presented as means ± SDs, and * and ** indicate P values of < 0.05 and < 0.01, respectively.(D) Transwell invasion assays of SR and MR cells treated with 1 μM PF06260933 or 10 nM GNE-495 for 24 h.

Figure 7 .
Figure 7. Schematic diagram of the suggested MAP4K4-ACSL4 signaling axis in radioresistant breast cancer cells.We suggest MAP4K4 positively regulates ACSL4 to mediate radioresistance by enhancing DNA damage response and inhibiting apoptosis.